PROJECT SUMMARY Immunotherapies for cancer, such as immune checkpoint inhibitors (ICI), have exhibited impressive efficacy for a broad spectrum of cancers, including melanoma. However, despite their potential to produce dramatic treatment responses, ICI have proven to be ineffective for most patients and tumor types. Consequently, there remains a need for new immunotherapies that are effective in patients who are resistant or non-responsive to current ICI. To this end, immune cytokines have potent immunostimulatory activities that make them attractive potential therapeutics. Compared to the effect of ICI to ?tune? existing immune responses, cytokines can tap into powerful programs that can effectively ?reprogram? immune cells and control their activation, differentiation, and proliferation. Among cytokines, Interleukin 18 (IL-18) is an appealing immunotherapeutic candidate given its ability to stimulate both adaptive and innate immunity through CD8+ T and NK cells, respectively. However, the activity of IL-18 is limited by an endogenous decoy receptor, IL-18BP, which is induced as part of a negative feedback loop by interferon gamma activity, reminiscient of the regulation of the PD-1:PD-L1 axis and suggesting that it may represent a ?soluble immune checkpoint? in cancer. Using directed evolution, the Ring lab has created an engineered version of IL-18 that is fully capable of activating the IL-18 receptor, but impervious to inhibition by IL-18BP. This decoy resistant IL-18 (DR-18) is active as a mono-immunotherapy in preclinical mouse models, where it can induce complete and durable tumor regressions. DR-18 also synergizes with ICI (anti-PD-1) therapy and is associated with high cure rates and limited toxicity in this setting. Notably, DR-18 is also effective for the treatment of MHC class I deficient murine tumors, which are resistant to ICI treatment, consistent with the ability of IL-18 to also activate anti-tumor activity of NK cells. In order to better understand the mechanism of DR-18 immunotherapy and enable its translation into the clinic, we propose to leverage a comprehensive series of sophisticated, genetically-defined syngeneic mouse models of melanoma called YUMM and YUMMER that we have developed. We propose to more comprehensively evaluate our hypothesis that bypass of the IL-18BP checkpoint by DR-18 will enable responses in ICI-resistant tumors in two Aims: 1) Evaluation and optimization of DR-18 therapy in melanoma, and 2) Development of DR-18 for the treatment of ICI-resistant tumors. We will utilize techniques including single-cell RNA sequencing and quantitiative pathology to better characterize DR-18 responses in the tumor microenvironment over time. Results from murine experiments will be functionally validated in fresh, dissociated human melanoma specimens. We aim to begin clinical trials with human DR-18 in 2020; these aims are focused on understanding and optimizing DR-18 in anticipation of this work.